Anti-Cariogenic Effect of A Cetylpyridinium Chloridecontaining Nanoemulsion

journal of dentistry 38 (2010) 742749
available at www.sciencedirect.com
journal homepage: www.intl.elsevierhealth.com/journals/jden
Anti-cariogenic effect of a cetylpyridinium chloridecontaining nanoemulsion

Valerie A. Lee *, Ramalingam Karthikeyan, H. Ralph Rawls, Bennett T. Amaechi
University of Texas Health Science Center at San Antonio, San Antonio, TX, United States
article info
abstract
Article history:
Objectives: The aim of this pilot study was to investigate the anticaries activity of a
Received 17 November 2009
nanoemulsion composed of soybean oil, water, Triton X-100 and cetylpyridinium chloride.
Received in revised form
Methods: Tooth blocks (3 mm length 3 mm width 2 mm thickness) were cut from
7 June 2010
smooth surfaces of selected molar teeth using a water-cooled diamond wire saw. The
Accepted 11 June 2010
blocks were randomly assigned to three experimental groups: (A) nanoemulsion, (B) 0.12%
chlorhexidine gluconate, and (C) no treatment. The formation of dental caries in human
tooth enamel was tested using a continuous flow dual-organism (Streptococcus mutans and
Keywords:
Lactobacillus casei), biofilm model, which acts as an artificial mouth and simulates the
Artificial caries
biological and physiological activities observed within the oral environment. Experimental
Nanoemulsion
groups A and B were treated with their respective solutions once daily for 30 s on each
Artificial mouth
occasion, while group C received no treatment. 10% sucrose was supplied every 6 h for 6 min
Transverse microradiography (TMR)
to simulate meals and pH cycling. The experiment lasted for 5 days, and the tooth blocks
were harvested and processed for demineralization assessment using transverse microradiography (TMR).
Results: For both lesion depth and mineral loss, statistical analysis indicated that Emulsion
was significantly lower than Control and Chlorhexidine, and Chlorhexidine was significantly lower than Control.
Conclusions: We conclude that cetylpyridinium-containing nanoemulsions appear to present a feasible means of preventing the occurrence of early caries.
# 2010 Elsevier Ltd. All rights reserved.
1.
Introduction
Biofilms are communities of microorganisms irreversibly

attached to a surface and containing an exopolymeric matrix.
Dental plaque, a well-recognized biofilm, is directly connected with the formation of dental caries. Despite the best
efforts of dental health professionals, dental caries are still
very prevalent in the general population. The average adult in
the U.S. has from 10 to 17 decayed, missing or filled permanent
teeth.1
Antimicrobial nanoemulsions are surfactant-containing oil

and water emulsions (droplet size 100300 nm) which have
been shown to be non-toxic to animals, but very effective
against many bacteria, viruses, fungi and spores in their freefloating or planktonic form.2 Preliminary morphological
studies suggest that nanoemulsions target bacterial cell
membranes.3 When growing as surface-adherent biofilms,
microorganisms undergo phenotypic changes that make them
very resistant to commonly used antimicrobial agents.4,5 As
many dental diseases, including dental caries, are associated
* Corresponding author at: 7703 Floyd Curl Dr., Comprehensive Dentistry, San Antonio, TX 78229-3900, United States.
Tel.: +1 210 567 3676; fax: +1 210 567 3669.
E-mail address: leev@uthscsa.edu (V.A. Lee).
0300-5712/$ see front matter # 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jdent.2010.06.001
with dental plaque, it is essential to test candidate antimicrobial agents against plaque biofilms, including cariogenic
biofilm.
The nanoemulsion employed in this work contains
cetylpyridinium chloride (CPC), a quaternary ammonium salt.
CPC is an effective antiplaque agent regulated by the Food and
Drug Administration. It has been proposed for inclusion in a
range of products for dental use, such as mouthrinses,
toothpastes, varnishes, orthodontic adhesives and liners for
glass ionomer cements.611 The safety and efficacy of CPC have
been evaluated extensively and proven based on cytotoxicity
data collected from many animal studies.1214
The antimicrobial efficacy of CPC-containing nanoemulsions against planktonic and biofilm bacteria, coupled with
low toxicity, makes this technology a candidate for control of
dental plaque and caries. The objective of this pilot study was
to determine if one nanoemulsion formulation effectively
prevented the formation of early caries associated with oral
biofilms. The null hypothesis was that CPC-containing
nanoemulsion would not protect enamel better against
demineralization compared to chlorhexidine gluconate, a
potent antiplaque agent.
2.
Materials and methods
2.1.
Preparation of nanoemulsion
A nanoemulsion was prepared in our laboratory in the

Division of Biomaterials at the University of Texas Health
Science Center at San Antonio using 25 vol.% soybean oil,
65 vol.% deionized water, 10 vol.% Triton X-100, 1 wt.%
cetylpyridinium chloride (SigmaAldrich, St. Louis, MO) and
a 25G reciprocating syringe. The ratio of ingredients closely
matches that described by Baker, inventor of the nanoemulsion, in his patent.2 Droplet size measurement was carried
[(Fig._1)TD$IG]
Fig. 1 Particle size analysis of a nanoemulsion composed

of 25 vol.% soybean oil, 65 vol.% deionized water, 10 vol.%
Triton X-100, 1 wt.% cetylpyridinium chloride. Mean
droplet size is 168 nm.
743
out using a dynamic light-scattering method (Protein Solutions, DynaPro, St. Paul, MN). The preparation method
resulted in a nanoemulsion with a narrow droplet size
distribution with a mean diameter of 168 nm (Fig. 1). The
nanoemulsion had the consistency and appearance of whole
milk.
2.2.
Preparation of teeth and experimental grouping
Human third molar teeth extracted due to impaction were

autoclaved (120 8C for 15 min), cleansed of soft tissue debris
and pumiced with non-fluoridated toothpaste slurry using a
Braun Oral-B Plaque Remover 3D electric toothbrush. The
teeth were examined by transillumination, and 13 teeth
without cracks, hypoplasia, white spot lesions and other
malformations were selected. Three tooth blocks (each 3 mm
length 3 mm width 2 mm thickness) were cut from the
smooth surfaces (buccal, lingual (palatal), mesial or distal) of
each tooth using a water-cooled diamond wire saw (Walter
Ebner, Switzerland). The blocks were randomly assigned to
three experimental groups, 13 blocks/group: (A) nanoemulsion, (B) 0.12% chlorhexidine gluconate (Peridex, 3M ESPE
Dental Products, St. Paul, MN), and (C) no treatment (Control).
The three groups were subjected to demineralization by
plaque growth in an artificial mouth described below to test
the ability of a CPC-containing nanoemulsion to inhibit the
formation of biofilms responsible for dental caries in human
tooth enamel.
2.3.
The artificial mouth system
This system is composed of multiple-station continuous flow

culture chambers (Fig. 2), which acts as an artificial mouth
and simulates the biological and physiological activities
observed within the oral environment. Each station consists
of a chamber bearing (i) a cylindrical clear-acrylic rod with
vertical grooves (CE) for mounting either whole tooth or
tooth blocks (I), (ii) a head assembly with two lines for supply
of simulated oral fluid (SOF), nutrients, experimental
reagents, and inoculation of the chamber with either singleor mixed-organism bacterial consortium (J and K), and (iii)
access for plaque sampling and electrode insertion for pH
monitoring. The SOF used in this system is BactoTM Todd
Hewitt broth (Fisher Scientific, Pittsburgh, PA) and was
adjusted to pH 7.0 (F). This was continuously circulated to
simulate saliva. Continuous circulation through the chambers at individually controlled flow rates (2 ml/min) via a
digital programmable pump (A) was maintained from a
reservoir (F). A complete circulatory system was established
by a return-flow line (H) from the chamber back into the
reservoir. The reservoir content was changed daily. The flow
rate of the SOF was varied in accordance with the oral
condition being simulated (e.g. stimulated or unstimulated
salivary flow). 10% sucrose was supplied every 6 h for 6 min to
simulate meals and pH cycling (G) using pump (B). All fluids
flowed uniformly as a thin film over the surface of the rod. The
entire assembly was housed inside a reach-in CO2 incubator
maintained at 5% CO2 and at a constant physiological
temperature of 37 8C. A micro-esophageal glass pH electrode
and a micro-reference electrode connected through a pH
744
[(Fig._2)TD$IG]
2.4.
Fig. 2 Schematic representation of the artificial mouth

system and its components. A, programmable circulating
pump for broth; B, programmable pump for broth and
sucrose; C, oral chamber for nanoemulsion; D, oral
chamber for chlorhexidine mouth rinse; E, oral chamber
for no treatment (Control); F, Todd Hewitt broth; G,
sucrose; H, return-flow line; I, tooth block; J, broth and
sucrose pumping tubes; K, broth circulating tube.
meter were installed in each chamber at the plaque growth

surface to monitor the intra-plaque pH. The completely
assembled system with reservoirs and contents were sterilized using ethylene oxide gas prior to each experiment.
Experimental procedure
The entire 5-day artificial mouth procedure is outlined in

Table 1. The three experimental groups were randomly
assigned to three culture chambers in the artificial mouth
system (13 blocks/chamber). Using heavy duty putty, these
blocks were embedded in the vertical grooves on the surface of
the cylindrical rod in the culture chamber. The blocks were
embedded such that their surfaces were flush with the surface
of the cylinder to permit streamlined flow of fluids, and the
exposed enamel was available for plaque growth and subsequent demineralization. Caries development on the experimental blocks was initiated by inoculation of the chambers by
1-h circulation of mixed Streptococcus mutans (NCTC 10449,
ATCC, Manassas, VA) and Lactobacilli casei (NCIB 8820, ATCC,
Manassas, VA) culture in Todd Hewitt broth (broth to
inoculum ratio 10:1) through the chambers. Inoculation was
repeated once daily for two consecutive days. Plaque biofilm
formation was confirmed by scanning the surface of two
blocks from each group using confocal microscopy on Day 5.
The system was then operated as described above by
continuous circulation of Todd Hewitt broth separately
through the three chambers to simulate saliva, and 10%
sucrose was supplied every 6 h for 6 min to simulate meals
and pH cycling. The pH of plaque in each chamber was
monitored at non-feeding time to check maintenance of
neutrality by CO2. All fluids, including inoculation, were
delivered at a flow rate of 2 ml/min. Change in plaque pH
following sucrose supply was monitored on two occasions on
the third day to confirm exhibition of Stephan-like curve of pH
fall under sucrose challenge. The chambers were accessed
individually from the top. The pH at non-feeding time
remained at approximately 6.8; following the supply of
sucrose it decreased gradually to as low as 5.2 and remained
at this level for the remaining part of the 6 min. Upon
withdrawal of sucrose and re-circulation of broth after 6 min,
the pH rose gradually to neutrality (6.8) after 45 min, but was
below 5.5 for 20 min. Experimental groups A and B were
treated with their respective solutions once daily for 30 s on
each occasion, while group C received no treatment. The
experiment lasted for 5 days, and the tooth blocks were
Table 1 Artificial mouth procedure.

Emulsion
Chlorhexidine
Control
1 h inoculation of bacterial consortium

30 s exposure to nanoemulsion
6 min exposure to 10% sucrose 3 daily

30 s exposure to chlorhexidine
Day 2


Day 3
Measurement of pH
Measurement of pH

Measurement of pH
Day 4
Day 5
Confocal microscopy
Confocal microscopy

Confocal microscopy
Day 1

harvested and processed for demineralization assessment

using transverse microradiography (TMR).
2.5.
Effect of nanoemulsion on biofilms
Since there was full growth of biofilm in group C, we decided to

use this group to test the effect of CPC-containing nanoemulsion on the viability of an existing biofilm.
Group C was exposed to emulsion for 30 s. Following this,
biofilm samples were collected from group C tooth slabs at 0
and 30 s, 1, 5, 10, 20, 30, 40, 50 and 60 min. The collected
samples were transferred to sterile polypropylene tubes
containing phosphate-buffered saline immediately following
collection and were dispersed by sonication (20 s) and vigorous
vortex mixing (30 s). Bacterial viability was assessed by serial
dilution, inoculation into culture media, and incubation at
37 8C and 5% CO2 for 2448 h.
2.6.
Transverse microradiography and image analysis
Three tooth slices (150 mm thick) were cut from each tooth
block using a water-cooled diamond wire saw (Buehler,
Switzerland). These slices were used to determine the
transverse microradiographic (TMR) parameters (mineral loss
(Dz) and lesion depth (LD)) of the caries lesion as follows. First,
both sides of the slice were polished using Adhesive Back 6 mm
lapping film in a MultiPrepTM Precision Polishing machine
(Allied High Tech, USA) to achieve planoparallel surfaces as
well as to reduce the thickness of the slice to 80 mm (the
appropriate thickness for TMR). Following this, the slices were
microradiographed on type lA high resolution glass X-ray
plates (Microchrome Technology, CA, USA) using a Phillips Xray generator system (Panalytical, Amsterdam) set up for this
purpose. The plates were exposed for 10 min at an anode
voltage of 20 kV and a tube current of 10 mA, and then
processed. Processing consisted of a 5 min development in
Kodak HR developer and 15 min fixation in Kodak Rapid-fixer
before a final 30 min wash period. After drying, the microradiographs were subjected to visualization with a Leica DMR
optical microscope linked via a Sony model XC-75CE CCTV
camera to a computer housing the image analysis program
(TMR2006 version 3.0.0.6, Inspektor Research, Amsterdam).
The
[(Fig._3)TD$IG] enhanced image of the microradiographs were analyzed
745
under standard conditions of light intensity and magnification

and processed, along with data from the image of the step
wedge, by the TMR program. The computer program calculated the parameter of integrated mineral loss (vol.% mm) and the
lesion depth (mm) based on the work described by de Josselin
de Jong et al.15 The integrated mineral loss was defined as the
difference in volume percent of mineral between sound and
demineralized tissue integrated over the lesion depth.16 The
lesion depth was assessed as the distance from the measured
sound enamel surface to the location in the lesion at which the
mineral content is greater than 95% of the mineral content in
sound enamel. By this method, the parameters of integrated
mineral loss (Dz, vol.% mm) and lesion depth (LD, mm) were
quantified for each caries lesion.
2.7.
Statistical procedures
Due to the small sample size of 13 teeth per treatment group,

box plots were used to depict the distributions of the lesion
depth and mineral loss data for treatment groups, for which
the lack of symmetry made the assumption of normality
inappropriate. As a result, non-parametric KruskalWallis
analyses of variance were performed for both measurements,
and non-parametric Spearman rank correlations between
lesion depth and mineral loss were performed for each
treatment group and all treatment groups combined. If a
KruskalWallis test was significant, then Bonferroni-adjusted
non-parametric MannWhitney U tests were performed
comparing Control vs. Emulsion, Control vs. Chlorhexidine,
and Emulsion vs. Chlorhexidine. For all statistical tests,
p < 0.05 was considered significant. Stata 11.0 (StataCorp,
College Station, TX) statistical software was used.
3.
Results
3.1.
Effect of treatments on demineralization
Both Emulsion and Chlorhexidine treatments correlated with

a reduction in lesion depth relative to Control, Fig. 3. Emulsion
treatment appeared to reduce lesion depth more than did
Chlorhexidine treatment. In an X-ray micrograph, loss of
mineral structure is noted as a darkened area. As seen in Fig. 3,
Fig. 3 Transverse microradiographic images from each experimental group. Lesion depth (mm), mineral loss (vol.% mm). (a)
21.1 mm, 250 vol.% mm; (b) 46.0 mm, 650 vol.% mm; (c) 70.8 mm, 1320 vol.% mm.
746
[(Fig._4)TD$IG]
[(Fig._6)TD$IG]
Fig. 4 Statistical analysis of lesion depth of the three

experimental groups. Emulsion was significantly lower
than Control and Chlorhexidine ( p < 0.001) and
Chlorhexidine was significantly lower than Control
( p = 0.001).
demineralization, beginning at the tooth surface and extending inward, occurred to a minimal extent in the Emulsion
group, and to progressively greater extents in the Chlorhexidine and Control groups. It is difficult to note differences in
mineral loss visually among the three groups of Fig. 3.
3.2.
Statistical results
Box plots for lesion depth for the three groups are presented in
Fig. 4 and percent mineral loss is presented in Fig. 5. The box
plot for lesion depth revealed a lack of symmetry about the
median for Emulsion and 3 low value outliers (represented as
dots) for Chlorhexidine and Control, so parametric statistical
analysis using the raw or log transformed data was not
appropriate. The KruskalWallis tests for lesion depth and
[(Fig._5)TD$IG]
Fig. 6 Agar plates showing growth of organisms collected

at 0 and 30 s after a 30 s exposure to emulsion. No growth
was found on plates of samples collected from 1 to 60 min
after exposure to CPC-containing nanoemulsion,
indicating the ability of the emulsion to disrupt existing
biofilms.
mineral loss were both significant ( p < 0.001), and subsequent

MannWhitney U tests for both measures indicated that
Emulsion was significantly lower than Control and Chlorhexidine ( p =0.001) and Chlorhexidine was significantly lower than
Control ( p = 0.001). The consistency in these results was
reflected by the overall Spearman correlation of 0.937 with 95%
confidence interval of (0.883, 0.967) between lesion depth and
mineral loss; within treatment groups, the rank correlations
were 0.928 with 95% c.i. (0.773, 0.979) for Emulsion, 0.797 with
95% c.i. (0.438, 0.937) for Chlorhexidine, and 0.556 with 95% c.i.
(0.008, 0.848) for Control.
3.3.
Microbial growth was observed only on plates of samples

collected at 0 and 30 s after exposure to CPC-containing
nanoemulsion, Fig. 6. No growth was observed on plates of
samples collected from 1 to 60 min after exposure, indicating
the efficacy of the emulsion in disrupting existing biofilms.
4.
Fig. 5 Statistical analysis of mineral loss of the three

experimental groups. Emulsion was significantly lower
than Control ( p < 0.001) and Chlorhexidine ( p = 0.001), and
Chlorhexidine was significantly lower than Control
( p < 0.001).
Effect of nanoemulsion on existing biofilms
Discussion
It is now well established that caries is dependent on

fermentable dietary carbohydrates. Cariogenic microorganisms, especially mutans streptococci, utilize dietary carbohydrate for their metabolic needs, creating organic acids as byproducts that demineralize the tooth tissue. A relatively high
proportion of mutans streptococci within dental plaque is
required for the initiation of caries, while lactobacilli are
associated with the progression of the carious lesion. The
organisms also utilize sucrose to synthesize and store both
extracellular polysaccharides and intracellular polysaccharides, leading to the production of biofilms.17
The artificial mouth model used in this study provided a
continuous or intermittent supply of nutrients to bacterial
747
plaque growing within an environment that mimics the in vivo

oral niches and habitats. The apparatus can be used to obtain
information on the etiology, prevention and management of
major oral diseases such as caries and possibly periodontal
diseases. Caries-like lesions of enamel, such as those created
in the artificial mouth, show all of the principal histological
features of natural caries and have been successfully used to
study the remineralization of enamel in vitro.18
Numerous studies have shown the marked resistance of
biofilms to antibiotics,1922 for reasons that are not fully
understood. One reason that biofilms are antibiotic resistant
may be the heterogeneity within the biofilm, with gradients of
pH, oxygen concentration, waste products and nutrients.2325
Antibiotics that require a certain environment to act may be
ineffective in certain areas of the biofilm. A second reason may
be related to the reduced growth rate of bacteria at the interior
of the biofilm. The most metabolically active organisms tend
to be located on the periphery of the biofilm.2628 There is a
general correlation between reduced growth rate and antibiotic resistance.2830 Bacteria in biofilms tend to undergo
phenotypic changes, and transfer of transmissible genetic
material occurs at an accelerated rate.4,5 Such changes may
increase the transfer of virulence and resistance factors to
antibiotics. In addition, it is thought that the matrix
surrounding the microcolonies serves as a protective barrier
to the intrusion of large molecules such as antibiotics.
The nature of the biofilm helps explain why dental caries
and periodontal diseases have been so difficult to prevent and
treat. Systemic and locally delivered antimicrobials have not
always proven successful, even when targeted at specific
microorganisms.31 Biofilms can be removed by mechanical
means, but they immediately begin to reform.
Antimicrobial nanoemulsions are surfactant-containing
oil-in-water emulsions (droplet size 100300 nm) which have
been shown to be non-toxic to animals but very effective
against many bacteria, viruses, fungi and spores in the freefloating or planktonic form.2,3,3235 The mechanism of action
of nanoemulsions is not fully understood, but it has been
proposed that the nanodroplets fuse with the outer membrane
of the microorganism, destabilizing the organisms lipid
envelope and initiating its disruption.36 The nanodroplets
acquire significant energy as they are formed by an extrusion
process under high shear forces, and may pass this energy to
the cell membrane upon contact. The nanoemulsions are
apparently non-toxic to skin and mucosal surfaces because of
the keratinized nature of the outer layers of those tissues.
Remarkably, the mechanism of action of nanoemulsions is
non-specific, unlike that of antibiotics, and is therefore very
unlikely to stimulate resistance as occurs with antibiotics.
Emulsions are thermodynamically unstable and tend to
revert to their unemulsified state. The emulsified state can be
prolonged considerably by the addition of a suitable surfactant, which resides at the interface between the oil droplets
and the continuous water phase. In our laboratory, we
conducted minimum inhibitory concentration assays
(MIC) every two weeks for one year using the CPC-containing
nanoemulsion from the present pilot study and planktonic S.
mutans. No change in MIC or in the appearance of
the emulsion was noted over the one-year interval (our
unpublished data).
The addition of a cationic halogen-containing compound

such as cetylpyridinium chloride (CPC) places a positive
surface charge on the nanodroplet by being incorporated as
a co-surfactant.2 Bacteria in dental plaque have a net negative
surface charge.37,38 In addition, exopolysaccharide chains,
which vary in size from 103 to 108 kDa, are usually negatively
charged, sometimes neutral or rarely positively charged.39
Positively charged nanodroplets should have increased potential to interact with the biofilm cells and matrix.
In addition to the above proposed action, quaternary
ammonium salts such as CPC have antimicrobial activity of
their own, apparently through multiple mechanisms. One
mechanism is thought to be due to disruption of intermolecular interactions, causing a dissociation of cellular membrane
lipid bilayers, compromising cellular permeability controls,
and inducing leakage of cellular contents.40 Longer exposure
times may result in additional breakdowns of intracellular
material which are indicative of autolysis.40,41 In addition, CPC
has an inhibitory action against fructosyltransferases, extracellular enzymes which synthesize fructans from sucrose,
which then play an important role in the progression of dental
caries by serving as an extracellular nutrition reservoir for
bacteria.42
Attempts to reduce or inhibit microbial adherence are a
viable means to control infection. The in vitro anti-adherence
of Candida albicans to oral buccal mucosal cells by low
concentrations of CPC has been described.43,44 Peripheral
coating of poly(ethylcyanoacrylate) nanoparticles has been
shown to result in decreased adhesion of C. albicans to buccal
epithelial cells in vitro.45 The mechanism of anti-adherence
appears to be both a disruption of the fungal membrane and a
steric interference of the approach of the microbial cell to the
epithelial cell. Similar mechanisms may operate in the vicinity
of the enamel surface.
In the present study, both Emulsion and Chlorhexidine
treatments were effective in resulting in reduced lesion depth
and mineral loss relative to controls, and Emulsion was more
effective than Chlorhexidine. The separate mechanisms of
action of both nanoemulsions and CPC may be operating to
reduce biofilm formation. It is possible that the positively
charged emulsion remains attached to the biofilm for a longer
time than does chlorhexidine and is therefore able to prevent
the formation of further biofilm.
In conclusion, CPC-containing nanoemulsions appear to
present a feasible means of preventing the occurrence of early
caries.
Acknowledgement
Supported by
K08DE018003.
NIH/NIDCR
T32
Grant
#DE14318
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Anti-Cariogenic Effect of A Cetylpyridinium Chloridecontaining Nanoemulsion

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Anti-Cariogenic Effect of A Cetylpyridinium Chloridecontaining Nanoemulsion

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journal of dentistry 38 (2010) 742749

journal homepage: www.intl.elsevierhealth.com/journals/jden